Proteins are involved in nearly every aspect of cellular function; therefore technologies for global protein characterization are necessary to virtually all areas of the biological sciences. Research in the field of proteomics has expanded tremendously over the last decade due to its potential to revolutionize biological and medical research, particularly for the development of new drugs, therapies and diagnostic methods. The term proteome is used to describe the entire set of proteins encoded by a genome. In a broader sense, however, the study of the proteome, referred to generally as the field of proteomics, involves the characterization of gene and cellular function by determining the identities, activities, interactions, localization and modifications of individual proteins and protein complexes present in a cell or tissue. Technology development has, and continues, to drive rapid evolution in this field.
Research has demonstrated that a proteome is typically characterized by very dynamic behavior. For example, the types of proteins expressed by a cell, as well as their abundances, post-translational modifications and subcellular locations, vary substantially with the physiological condition of a cell or tissue, including the onset and progression of disease. Accordingly, quantitative characterization of changes in protein content, composition and activity at the organ, tissue, and cellular levels provide information useful for identifying new biological targets for drug development and novel biomarkers for the diagnosis and early detection of disease. Furthermore, proteomics research is highly complementary to other functional approaches for understanding cellular and sub-cellular processes, such as microarray-based expression profiles, systems level genetics, and small molecule based arrays.
The complexity of proteomics, at least in part, is due to the large number of proteins and protein complexes corresponding to a genome. For example, the human proteome is expected to consist of between about 400,000 to about 1,000,000 proteins, which interact to form a huge number of protein-protein complexes important in regulating cellular behavior. The complexity of the human proteome is significantly compounded by the large dynamic range observed for protein expression, typically exceeding over six orders of magnitude, and by post-translational modifications that critically impact protein activity and function. To address this inherent complexity, a number of high throughput platforms for identifying and characterizing proteins have been developed, including two-dimensional gel electrophoresis (2D-GE) protein identification methods, genetic readout experiments, such as the yeast two-hybrid assay, micro-array and chip technologies, and mass spectrometry methods.
Mass spectrometry has emerged as a preferred experimental tool for proteomics given its sensitivity, speed, versatility and specificity. [McLafferty, F. W.; Fridriksson, E. K.; Horn, D. M.; Lewis, M. A.; Zubarev, R. A., Science 1999, 284, 1289-1290]. In addition, mass spectrometric analysis is well suited for automated, high throughput operation, particularly when combined with multidimensional separation techniques, such as high performance liquid chromatography (HPLC) or capillary electrophoresis. As a result significant research has been directed toward developing mass spectrometry-based techniques for identifying proteins in complex mixtures, probing protein-protein interactions and characterizing post-translational modifications. The application of mass spectrometric methods to protein identification has been the subject of numerous scientific publications including “Mass Spectrometry and the Age of the Proteome,” Yates, J. R., J. Mass Spectrometry, Vol 33, 1-19 (1998); “Mass Spectrometry-based Proteomics,” Aebersold, R., Mann, Matthias, Nature, 2003, 198-207; “Proteomics to Study Genes and Genomes,” Pandey, A. and Mann, M., Nature, 2000, 405, 837-846; “Mass Spectrometry in Proteomics,” Aebersold, R. and Goodlett, D. R., Chem. Rev., Vol 101, 269-295 (2001); “An automated Multidimensional Protein Identification Technology for Shotgun Proteomics,” Wolters, D. A., Washburn, M. P. and Yates, J. R., III, Anal Chem., Vol 73, 5683-5690 (2001); and “Analysis of Proteins and Proteomes by Mass Spectrometry,” Mann, M., Hendrickson, R. C. and Pandey, A., Annu. Rev, Biochem, Vol. 70, 437-473 (2001), which are all hereby incorporated by reference in their entireties to the extent not inconsistent with the present description.
Traditionally, protein sequences are determined by stepwise enzymatic degradation of purified proteins into peptide fragments, for example by trypsin digestion, subsequent analysis of the peptide fragments by mass spectrometry and protein/peptide characterization using advanced bioinformatic tools. Recent interest has focused on the characterization of proteins with complex post-translational modifications, as these modifications are known to play a critical role in the function and regulation of proteins. [Ge, Y.; Lawhorn, B. G.; EINaggar, M.; Strauss, E.; Park, J. H.; Begley, T. P.; McLafferty, F. W., J. Am. Chem. Soc. 2002, 124, 672-678. Ge, Y.; EINaggar, M.; Sze, S. K.; Bin Oh, H.; Begley, T. P.; McLafferty, F. W.; Boshoff, H.; Barry, C. E., J. Am. Soc. Mass Spectrom. 2003, 14, 253-261. Sze, S. K.; Ge, Y.; Oh, H.; McLafferty, F. W., Proc. Nat'l Acad. Sci. USA 2002, 99, 1774-1779.]. Reversible protein phosphorylation, for example, is a ubiquitous post-translational modification involved in many biological processes, including cell growth, division, and signaling. [Hunter, T., Cell 2000, 100, 113-127]. In addition, aberrant protein phosphorylation is indentified as responsible for many human diseases, including cancer and heart diseases. As a result of the well recognized importance of protein phosphorylation, considerable proteomic research is directed to the identification of phosphorylation states and specific phosphorylation sites of phosphoproteins/phosphopeptides.
Advanced mass spectrometry methodologies have been developed enabling selective identification and characterization of the phosphorylation state of single proteins or peptides (See. e.g. Shi, S. D. H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W., Anal. Chem. 2001, 73, 19-22; Carr, S. A.; Huddleston, M. J.; Annan, R. S., Anal. Biochem. 1996, 239, 180-192; Kjeldsen, F.; Savitski, M. M.; Nielsen, M. L.; Shi, L.; Zubarev, R. A., Analyst 2007, 132, 768-776; Steen, H.; Jebanathirajah, J. A.; Rush, J.; Morrice, N.; Kirschner, M. W., Mol. Cell. Proteomics 2006, 5, 172-181. Zabrouskov, V.; Ge, Y.; Schwartz, J.; Walker, J. W., Mol. Cell. Proteomics 2008, 7, 1838-1849. Ge, Y.; Rybakova, I.; Xu, Q.; Moss, R. L. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 12658-12663.). Implementation of these techniques, however, presents significant challenges given the low abundance of phosphoproteins and substoichiometric phosphorylation commonly observed in many biological environments. To realize the benefits provided by mass spectrometry-based methods for proteomics, therefore, techniques for purifying and enriching phosphoproteins and phosphopeptides are currently needed. A number of either affinity- or chemical derivatization-based purification and enrichment techniques have attracted attention recently to complement mass spectrometry based proteomics analysis of phosphorylated proteins and peptides [See, e.g., “Reproducible isolation of distinct, overlapping segments of the phosphoproteome,” Bodenmeiller, B., Mueller, L. N., Mueller, M., Domon, B., Aebersold, R., Nature Methods, 2007, 4, 231-237; “Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry”; Tao, W. A., Wollscheid, B., O′Brien, R., Eng, J. K., Li, X. J., Bodenmiller, B., Watts, J. D., Hood, L. and Aebersold, R., Nature Methods, 2005, 591-598; and “Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome”; Oda, Y., Nagasu, T. and Chait, B. T., Nature Biotechnol., 2001, 379-382, which are all hereby incorporated by reference in their entireties to the extent not inconsistent with the present description].
In contrast to the chemical derivatization-based method which suffers from side reaction, increased sample complexity and potential loss of phosphate, the affinity based method is more commonly used. Affinity based methods for phosphopeptide enrichment using immobilized metal ion affinity chromatography (IMAC) are well developed for mass spectrometry-based proteomic analysis. [See, e.g., Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G., Nature 1975, 258, 598-599; and Zhang, X.; Ye, J. Y.; Jensen, O. N.; Roepstorff, P., Mol. Cell. Proteomics 2007, 6, 2032-2042, which are all hereby incorporated by reference in their entireties to the extent not inconsistent with the present description]. Conventional techniques include use of metal ions, such as Ga(III) and Fe(III), that provide specific and reversible chemisorption of phosphate groups of phosphoproteins and phosphopeptides. Such metal ion affinity materials, however, are known to also bind to certain non-phosphorylated amino acid residues, such as glutamic and aspartic acid, degrading overall specificity. An alternate approach uses metal oxide materials for affinity based enrichment of phosphopeptides. TiO2 and ZrO2, metal oxides, for example, have been pursued and are believed to exhibit less non-specific binding and higher specificity for trapping phosphates as compared to conventional metal ion affinity materials. [See, e.g., Pinkse, M. W. H.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R., Anal. Chem. 2004, 76, 3935-3943. Kweon, H. K.; Hakansson, K., Anal. Chem. 2006, 78, 1743-1749]. Nanoparticle metal ion and metal oxide materials have also been explored due to their expected higher capacities than microparticles. [See, e.g., Chen, C. T.; Chen, Y. C., J. Biomed. Nanotechnol. 2008, 4, 73-79; Zhou, H. J.; Tian, R. J.; Ye, M. L.; Xu, S. Y.; Feng, S.; Pan, C. S.; Jiang, X. G.; Li, X.; Zou, H. F., Electrophoresis 2007, 28, 2201-2215].
U.S. Patent Application Publication No. 2006/0014234, published on Jan. 19, 2006, discloses a chemical derivatization-based method for the enrichment and separation of phosphorylated peptides or proteins. Techniques disclosed in this reference include the use of an organo-functionalized separation medium for enriching phosphorylated peptides and proteins present in complex mixtures. Separation media functionalized with diazo moieties linked by an organic group are reported as particularly useful for mass spectrometry based characterization of phosphopeptides. This reference further discloses enrichment processes involving protection of carboxylate groups to reduce interferences with selective immobilization of phosphorylated peptides. A wide range of organo-functionalized separation materials are disclosed in this reference including resins, polymers, metal oxides, metal hydroxides, mesoporous materials, metals, silicate clays, and metal phosphates.
It will be appreciated from the foregoing that there is currently a need for improved methods and materials for identifying and analyzing phosphorylated peptides and proteins.